PROJECT SUMMARY/ABSTRACT Prostate MRI is becoming increasingly critical in prostate cancer detection, diagnosis, and treatment algo- rithms. However, its sensitivity and specificity remain suboptimal. Within the multiparametric paradigm of pros- tate MRI, the contrast enhanced imaging component utilizing existing clinical contrast agents adds relatively little if any increased sensitivity to the examination. We see our long term goal to develop MRI contrast agents targeted to prostate cancer, as representing the largest potential opportunity for improving prostate MRI. The overall objective of this proposal, is to create and validate novel activatable aggregating MRI contrast agents specifically targeted to prostate specific membrane antigen (PSMA). Our first innovative strategy in accomplishing our goal is to focus on developing MRI contrast agents that can activate and accumulate in the area of pathology. Targeted MRI contrast agents whose accumulation within the body is based on single contrast agent-target interactions are in general unrealistic, as with rare exceptions biological target densities are inadequate to support the accumulation of sufficient contrast agent to generate a reliably detectable signal increase with MRI. We have previously developed activatable contrast agents whose accumulation in the setting of cell death (apoptosis) is amplified by enzymatic action. We will build off this prior work to now develop contrast agents whose accumulation can be amplified in the setting of prostate cancer. Our second innovative strategy is to harness the extracellular enzymatic activity of prostate specific membrane antigen (PSMA) to provide this amplification. PSMA is an attractive molecular imaging target as it is reliably overexpressed in prostate cancer. We will design our contrast agents such that after being cleaved by PSMA, these agents will accumulate in prostate cancer either by aggregating outside or inside of the cells of interest. Our third innovative strategy is to couple MRI contrast agent development with validation in a prostate cancer patient derived xenograft (PDX) model. Proper validation of molecular imaging agents is critical for achieving robust experimental results that support future translation of these agents to clinical use. Prior work has often been overly reliant on conventional xenograft models that are poor models of cancer as it occurs in humans. The significance of the proposed research is that it demonstrates a generalizable mechanism applicable to ex- tracellular enzymatic targets for overcoming sensitivity limitations of targeted MRI contrast agents. Such work has the potential to improve the sensitivity of prostate MRI, and through this improve detection and eventually treatment of prostate cancer.